The present invention relates to procedures for treating biological specimens to make their nucleic acids available for various purposes, such as nucleic acid hybridization assays for the diagnosis of disease and other purposes, and for amplification of nucleic acids by the polymerase chain reaction (PCR) or other target amplification procedures. Specifically, the present invention relates to convenient procedures for making nucleic acids available that prevent degradation of the nucleic acids by endogenous nucleases present in the biological sample.
Many diagnostic procedures are based on detection of specific nucleic acid (DNA or RNA) sequences present in a biological sample. For example, the sample may contain bacteria, viruses, or other microorganisms whose presence must be ascertained to determine the cause of an infectious disease. In other instances, the nucleic acid sequence may be sought within the DNA of a human white blood cell in order to establish the presence of a mutation associated with cancer or a genetic disease.
For such a diagnostic analyses, it is necessary to make available the specific nucleic acid that may be present in the sample. Frequently, the nucleic acid will be contained within a bacterium, fungus, virus, or other microorganism or within human cells such as white blood cells. It may further be contained within other structures such as ribosomes, plasmids, or chromosomal DNA. In order to perform hybridization reactions to detect specific nucleic acids or to amplify them using PCR or other target amplification methods, the nucleic acid must be released from these organisms and/or structures.
Unfortunately, such release exposes the nucleic acids to degradation by endogenous nucleases present in the sample, which may exist in such abundance that the nucleic acid is almost instantaneously destroyed.
The problem is particularly acute when the specific nucleic acid is an RNA, since RNAses are abundant in most biological samples and are often extremely resistant to treatments that readily inactivate many other enzymes.
To deal with this problem, it is common in the art to employ a variety of means to purify the nucleic acids from the biological sample. For example, anionic detergents and chaotropic agents such as guanidinium salts have been used to simultaneously inactivate or inhibit nuclease activities and release nucleic acids from within cells and subcellular structures. Unfortunately, these agents are also potent inhibitors of the enzymes used in target amplification processes or in many hybridization detection methods or, in the case of chaotropes, may interfere with hybridization itself. Therefore, it has been necessary to use additional steps to remove these agents and recover the nucleic acids.
The most commonly used procedure is to precipitate the nucleic acids from the sample using various salts and ethanol. The sample must be kept at reduced temperature (usually xe2x88x9220xc2x0 C. or lower) for some hours and centrifuged at high speed in order to achieve good yields of nucleic acids in most instances.
Because other macromolecules also precipitate under these conditions producing a sticky, intractable mass that entraps the nucleic acids, it has been frequently necessary to resort to extraction of the sample with hazardous organic solvent mixtures containing phenol, cresol, and/or chloroform prior to ethanol precipitation. In some cases when anionic detergents are used, proteases that are active in the presence of these detergents, such as proteinase K or pronase, are used to partially degrade protein components of the sample to minimize entrapment during organic solvent extraction, and/or degrade components that may not be extracted by the solvent treatment.
It will be readily appreciated that these methods are complex, tedious, labor-intensive, and slow. If great care is not taken in performing the procedure, residual contamination with nucleases can occur, and the sample nucleic acids will be degraded or lost. Diagnostic tests performed with such samples may give false negative results. False negative results can also be obtained if residual anionic detergents, chaotropic salts, or ethanol remain in the sample and inhibit hybridization and/or target amplification procedures. If anionic detergents and proteases have been used, residual proteolytic activity can also degrade the enzymes used in target amplification and/hybridization detection reactions and produce false negative results. On the other hand, improper processing with these methods can also result in the isolation of denatured proteins or other macromolecules that can entrap labelled probes and produce false positive results with diagnostic tests involving nucleic acid hybridization. Thus, these procedures are not well suited for routine processing of biological specimens received in clinical laboratories in any quantity.
Particularly, trouble is encountered with many biological samples in which the desired nucleic acid species is RNA, and the sample contains significant amounts of RNAse of the xe2x80x9cpancreaticxe2x80x9d type (also frequently referred to as xe2x80x9cribonuclease Axe2x80x9d). Pancreatic RNAses are present in serum and plasma and in many tissues of the body. They are resistant to denaturation by heat and acids and will even withstand boiling in 1 N HCl for 10 minutes without loss of activity. They are inhibited by anionic detergents, chaotropes, and organic solvents such as phenol, but are not irreversibly inactivated by these agents; therefore, when the detergents, chaotropes, or solvents are removed, the RNAse (if not eliminated by careful extraction) can proceed to degrade the desired RNA.
Exposure to strong alkali will irreversibly inactive these RNAses; however, such conditions also result in the degradation of RNA itself.
The present invention addresses these problems by providing a method for conveniently inhibiting and inactivating nucleases in biological samples while making available sample nucleic acids for hybridization assays, target amplification procedures, or other uses. Inhibitory detergents or chaotropes are not required in the sample, and there is no residual proteolytic activity. The method is simple and applicable to processing large numbers of samples simultaneously. Unlike ethanol precipitation methods, it does not use hazardous organic solvents, nor require equipment for cooling the sample or recovering precipitates by centrifugation.
The present invention features a procedure for irreversibly inactivating endogenous nucleases in biological samples by reducing the pH below that at which the endogenous nucleases present in the sample are active, adding a protease which is active at that pH and which degrades any nucleases that have not been irreversibly inactivated by exposure to low pH, and then inactivating the protease (after it has done its work) by raising the pH. At the higher pH, the chosen protease is either inactive or is irreversibly inactivated. If possible, the protease is chosen so as to aid in the digestion of other macromolecules in the sample that may interfere with the intended use of the sample, and chosen to help make available the desired nucleic acids by degrading microorganism cell walls, virus particles, ribosomes, and/or other structures containing the desired nucleic acids. Alternatively, solubilization of these structures and release of the nucleic acids may be effected by the use of detergents, heat, or other means once sample nuclease activity has been effectively controlled, reduced, or eliminated.
In general, the biological sample is adjusted to a low pH where the endogenous nucleases are either irreversibly inactivated or are effectively inhibited. Exogenous acid protease (such as pepsin) is then added or may be added simultaneously with the pH lowering solution. The action of the acid protease digests the endogenous nucleases present in the sample and irreversibly inactivates them so that they will not degrade the sample nucleic acids when the pH is subsequently raised. In addition, the protease will usually act to liberate the nucleic acids from microorganisms, human cells, or subcellular components such as ribosomes and nuclei. It will also degrade many protein components of the biological sample, including ones that may interfere with subsequent use of the sample for hybridization assays and/or target amplification procedures.
The acid protease selected should have activity at an acidic pH, pH 1.0 to pH 4.0, and be able to digest a wide variety of proteins, including the nucleases found in the sample as well as other unwanted components. In addition, it should ideally be able to digest components which contain the desired nucleic acid. In some cases, it may be desirable to select a protease of more limited specificity in order that a nucleic acid (whose presence in free form in the sample is undesirable) is not liberated from its component structures.
The acid protease should also be checked to ensure that it is itself free of nuclease activities that are active at the chosen acidic pH or which are resistant to degradation and inactivation by the chosen acidic protease.
Commercial preparations of acid proteases may be contaminated with such enzymes and should be purified, if necessary, to eliminate them. In the examples that follow, a procedure for purifying commercially available pepsin preparations to eliminate residual RNAse activities that are not eliminated by pepsin digestion is given. Equivalent procedures can be used for other proteases.
The protease is rendered inactive by the simple act of raising the pH. With some acid proteases, this is sufficient to completely stop further proteolytic digestions and may irreversibly denature and inactivate the enzyme. With other proteases, it may be necessary to resort to heating the sample to achieve complete inactivation of the protease. Since the nucleases have been destroyed and nucleic acids are not damaged by brief exposure to heat at neutral pH, they will survive this procedure intact.
Accordingly, this invention provides a simple procedure to extract nucleic acids in vitro. This procedure can be used for processing many biological samples, including those containing viruses, such as hepatitis C virus, which presents particular difficulties because it is an RNA-containing virus which is difficult to open, the sample is serum or plasma which contains significant amounts of pancreatic-type RNAse activity, and the virus is often present in very low amounts which makes recovery of the nucleic acids by ethanol precipitation techniques difficult.
Thus, in a first aspect, the invention features a method for purifying or making available a nucleic acid from a biological sample by acidifying the biological sample to a pH at which endogenous nucleases (capable of degrading the desired nucleic acids) are less active, e.g., to a pH between 1.0 and 4.0; contacting the biological sample with an exogenous acid enzyme active at that pH; incubating the sample until endogenous nucleases have been degraded to insignificant levels (i.e., to a level where their effect on levels of nucleic acids in the sample is insignificant, e.g., at a level where less than 5% of the nucleic acids are degraded over a period of 60 minutes at 37xc2x0 C. in a standard salt solution); and raising the pH of the biological sample with a base to a pH sufficient to render the exogenous protease less active, e.g., to a pH at which the protease is no longer active.
By xe2x80x9cmaking availablexe2x80x9d is meant that the nucleic acid is accessible for later analyses, such as hybridization or amplification.
By xe2x80x9cless activexe2x80x9d is meant, with respect to endogenous nucleases, is less nuclease activity than prior to treatment (under the same conditions). By xe2x80x9cless activexe2x80x9d is meant, with respect to exogenous proteases, is less protease activity than prior to treatment. Preferably, the lower exogenous protease activity is insufficient to reduce the activity of enzymes used in later processes involving the isolated nucleic acids.
By xe2x80x9cdegradedxe2x80x9d is meant that the activity of the nucleases is reduced to a level which will allow later experiments or manipulations of the isolated nucleic acids.
By xe2x80x9cdesired nucleic acidxe2x80x9d is meant a nucleic acid that is obtained, possibly along with other nucleic acids by this invention, and can subsequently be specifically identified.
In preferred embodiments, a biological sample is chosen from tissue cells, blood components, and other human biological materials which may contain infectious disease agents; the biological sample consists of human white blood cells, cancer cells, or other cells which offer a convenient source of human cellular nucleic acid for genetic analysis, body fluids, secretions, or tissues; the acid protease is pepsin; the pH of the sample after incubating, preferably at pH 4 or lower in the presence of exogenous protease, is raised up to a level suitable for subsequent use, but below that level at which the exogenous protease is completely inhibited or inactivated; in the acidifying step the pH is adjusted to between 1.0 and 4.0; in the raising step the pH is adjusted to be greater than 6.0; following the raising step the sample is heated to aid inactivation of the acidic protease and/or other enzyme activities present in the sample; detergents are added to the sample to aid release of the desired nucleic acid from other sample components; and the time of the incubating is longer than necessary to reduce endogenous nuclease levels to insignificant levels, in order to effect lysis of sample components and/or degradation of other sample components.
In related aspects, the invention features a kit including components necessary to carry out the method of this invention, and a method for purifying pepsin from RNAses for use in this method. Such purification makes use of an RNAse adsorbent which does not adsorb proteases, e.g., Macaloid or bentonite. Macaloid is a natural clay mineral product that has the property of adsorbing RNAses to its surface. Bentonite is a similar material which is a colloidal native hydrated aluminum silicate clay consisting primarily of montmorillonite. Both can often be used interchangeably to remove RNAses from a variety of biological materials. Other more-or-less specific adsorbents could be used provided they adsorb the RNAses and not the desired protease.
The present invention provides a procedure for isolating nucleic acids from different types of biological samples under acidic conditions where the degradation of these nucleic acids is minimized. This process is particularly useful for obtaining nucleic acids from specimens where there is a risk of significant degradation of nucleic acids by endogenous nucleases. Nucleic acids which can be isolated by this procedure include naturally occurring nucleic acids and synthetic nucleic acids or oligonucleotides.
The biological samples containing the nucleic acids to be isolated include tissue cells, blood components, viruses, microorganisms, pathogenic organisms, and body fluids containing these various organisms.
An initial step of the procedure of the present invention is adjustment of the acidity of the biological specimen containing the desired nucleic acids to about pH 4 or lower. At this pH, nucleases which may be present in the biological sample are not active. KClxe2x80x94HCl buffer, glycine-HCl buffer, acetic acid buffer and various other acidic buffer compositions having buffering activity in acidic conditions, can be used to reduce the pH of the specimen. Endogenous nucleases present in clinical samples do not work (i.e., have negligible enzymatic activity) at a sufficiently acidic pH, since this pH is far below their optimum range. For example, serum RNAse has its optimum pH at about 6.5. It has almost no activity at pH 3.0 or lower. Leukocyte RNAse has an optimum pH range from 6.0 to 6.5 and has virtually no enzymatic activity at pH 4.0 or lower. Therefore, the adjustment of acidity of the mixture to about pH 4 or lower will prevent the action of most known endogenous RNAses.
Similarly, serum deoxyribonuclease activity has its optimum pH at about 5.8 to 7.0, depending upon the type of divalent metal(s) present. It shows little activity below pH 5.0, regardless of metal ion present. Leukocyte DNAse has an optimum pH range from about 4.0 to about 5.0, and it is virtually inactive below pH 3.0. The exact pH ranges at which nucleases found in clinical samples are active will depend upon such variables as the type of buffer used to control the pH, metal ion requirements, if any, and temperature.
For use of the present invention, those skilled in the art know how to assay for activities that degrade one or more nucleic acids of interest, and can easily determine the appropriate pH, buffer, and temperature that is needed for a particular sample type. In particular, it may be important to lower the temperature and minimize the time of exposure to very low pH when it is desired to recover DNA, since depurination of the DNA can occur at low pH when higher temperatures and longer times are employed. However, it is an important feature of the present invention that some DNA depurination and chain breakage may occur and is useful in that it helps to break up gelatinous aggregations of DNA that are produced when some biological specimens (for example, white blood cell pellets) are lysed. Thus, the present invention can address this additional specimen processing problem as an added benefit of the method.
The next step (which can be performed simultaneously, or even before, the first step if desired) is addition of an acidic protease into the acidified biolohical samples. The endogenous nucleases in the reaction mixture are digested and irrversibly inactivated by this protease. In this step, the desired nucleic acids may also be liberated from the biological sample into the aqueous solution when the biological components, e.g., cell membranes, are also digested by the acidic protease. Pepsin is one example of an acidic protease which can be used in this step. Other proteases can be used as long as they retain enzymatic activities under acidic conditions that inactivate the unwanted nuclease activities present in the biological sample. Such proteases are readily identified by those in the art using standard procedures.
Nucleic acids released by the steps described above are stable because the aqueous solution no longer contains active nuclease (even after neutralization of the solution to inactivate the acidic protease by addition of alkali). Such neutralization provides physiological conditions suitable for subsequent enzymatic reactions, e.g., for nucleic acid amplification procedures such as PCR, and cDNA polymerization methods. Thus, the neutralized solution may be used directly in such procedures without further processing, e.g., without removal of strong anionic detergents or other harsh agents which may affect the activity of enzymes used in subsequent processes.
This procedure provides significant advantages over other nucleic acid isolation methods, since no process is required to remove guanidine isothiocyanate or other denaturing agents (used in other procedures). The exogenous acid protease inactivates endogenous nucleases irreversibly and liberates nucleic acids from the biological sample in one step. This procedure may be readily and simply used to isolate nucleic acids from a biological sample for genetic diagnosis and thus is useful in a clinical laboratory.
The following examples are set forth to illustrate various aspects of the present invention, but do not limit in any way its scope as more particularly set forth in the claims.